The Lys-Asp-Tyr Triad within the Mite Allergen Der p 1 Propeptide Is a Critical Structural Element for the pH-Dependent Initiation of the Protease Maturation.

The major house dust mite allergen, Der p 1, is a papain-like cysteine protease expressed as an inactive precursor, proDer p 1, carrying an N-terminal propeptide with a unique structure. The maturation of the zymogen into an enzymatically-active form of Der p 1 is a multistep autocatalytic process initiated under acidic conditions through conformational changes of the propeptide, leading to the loss of its inhibitory ability and its subsequent gradual cleavage. The aims of this study were to characterize the residues present in the Der p 1 propeptide involved in the initiation of the zymogen maturation process, but also to assess the impact of acidic pH on the propeptide structure, the activity of Der p 1 and the fate of the propeptide. Using various complementary enzymatic and structural approaches, we demonstrated that a structural triad K17p-D51p-Y19p within the N-terminal domain of the propeptide is essential for its stabilization and the sensing of pH changes. Particularly, the protonation of D51p under acidic conditions unfolds the propeptide through disruption of the K17p-D51p salt bridge, reduces its inhibition capacity and unmasks the buried residues K17p and Y19p constituting the first maturation cleavage site of the zymogen. Our results also evidenced that this triad acts in a cooperative manner with other propeptide pH-responsive elements, including residues E56p and E80p, to promote the propeptide unfolding and/or to facilitate its proteolysis. Furthermore, we showed that acidic conditions modify Der p 1 proteolytic specificity and confirmed that the formation of the first intermediate represents the limiting step of the in vitro Der p 1 maturation process. Altogether, our results provide new insights into the early events of the mechanism of proDer p 1 maturation and identify a unique structural triad acting as a stabilizing and a pH-sensing regulatory element.

Plant phenolics are detoxified by prophenoloxidase in the insect gut.

Plant phenolics are a group of important secondary metabolites that are toxic to many animals and insects if ingested at high concentrations. Because most insects consume plant phenolics daily, they have likely evolved the capacity to detoxify these compounds. Here, we used Drosophila melanogaster, Bombyx mori and Helicoverpa armigera as models to study the metabolism of plant phenolics by prophenoloxidases. We found that insect foreguts release prophenoloxidases into the lumen, and that the survival of prophenoloxidase-deletion mutants was impaired when fed several plant phenolics and tea extracts. Using l-DOPA as a model substrate, biochemical assays in large Lepidopteran insects demonstrated that low levels of l-DOPA are rapidly metabolized into intermediates by phenoloxidases. Feeding with excess l-DOPA showed that the metabolic intermediate 5,6-dihydroxyindole reached the hindgut either by passing directly through the midgut, or by transport through the hemolymph. In the hindgut, 5,6-dihydroxyindole was further oxidized by prophenoloxidases. Intermediates exerted no toxicity in the hemocoel or midgut. These results show that plant phenolics are not toxic to insects unless prophenoloxidase genes are lost or the levels of phenolics exceed the catalytic activity of the gut prophenoloxidases.

Zingiber cassumunar ROXb. and its active constituent inhibit MMP-9 direct activation by house dust mite allergens and MMP-9 expression in PMA-stimulated human airway epithelial cells.

BACKGROUND: House dust mite (HDM) induced matrix metalloproteinase (MMP)-9 plays a role in asthma. Zingiber cassumunar Roxb. (Phlai in Thai) has been used in folk medicine for asthma treatment. OBJECTIVE: We investigated effects of Phlai and its constituent (E)-4-(3',4'-dimethoxyphenyl)but-3-en-1-ol (compound D) on the cleavage of pro- MMP-9 by HDM. The effects of these compounds on phorbol 12-myristate 13-acetate (PMA)- induced MMP-9 gene and protein expression in airway epithelial cells (NCI-H292) were also investigated. METHODS: Pro-MMP-9 was directly activated in vitro with HDM in the presence or absence of the ethanolic extracts of Phlai or compound D for 1 hour. The amount of activated MMP-9 was determined using gelatin zymography. To study the cellular response of Phlai, NCI-H292 cells were pretreated with crude Phlai extracts or compound D for 2 hours, and then the cells were stimulated with PMA for 48 hours. The mRNA RT-PCR and Western blotting, respectively. MMP-9 activity was determined by gelatin zymography. RESULTS: Crude Phlai extracts (0.25 - 2.0 mg/ml) and compound D (0.5 - 4.0 mg/ml) inhibited pro- MMP-9 cleavage by HDM. Furthermore, crude Phlai extracts (100 mg/ml) and compound D, at concentrations of 50 and 100 mg/ml, attenuated the PMA-induced MMP-9 gene and expression in NCI-H292 cells. These compound also suppressed MMP-9 release from PMA-induced NCI-H292 cells. CONCLUSION: The crude ethanolic extract of Z. cassumunar and its active constituent compound D inhibited the cleavage of pro-MMP-9 by HDM. They also inhibited PMA-induced MMP-9 gene and protein synthesis in human airway epithelial cells.

The roles of Na⁺/K⁺-ATPase α-subunit gene from the ridgetail white prawn Exopalaemon carinicauda in response to salinity stresses.

Na(+)/K(+)-ATPase (NAK) is one important transporter protein and plays a key role in maintaining osmotic homeostasis in low and high salinity acclimation in variety of crustacean species. The ridgetail white prawn Exopalaemon carinicauda is an euryhaline and economic shrimp species in China, but it remains unclear about its mechanism of salinity adaption. In this study, a full-length of Na(+)/K(+)-ATPase α-subunit (α-NAK) cDNA was cloned from E. carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of α-NAK was of 3680 bp, containing an open reading frame (ORF) of 3030 bp encoding a polypeptide of 1009 amino acids with the predicted molecular weight of 112.27 kDa. Eight transmembrane domains and two sites of phosphorylation and ATP binding were identified in E. carinicauda α-NAK. BLAST analysis revealed that the sequence of α-NAK amino acids of E. carinicauda shared more than 75% homologies with those of other crustacean. Real time quantitative RT-PCR analysis indicated that E. carinicauda α-NAK gene could be detected in all the tested tissues with highest expression level in gill. The expression profiles of E. carinicauda α-NAK transcripts were analyzed in gill and hepatopancreas tissues after salinity stresses. The results showed that the expression level of E. carinicauda α-NAK gene in both gill and hepatopancreas reached peak at different time after low and high salinity stresses, and showed different expression profiles. The expression profiles of proPO transcripts in gills after salinity stresses also indicated α-NAK and proPO played synergistic actions for salinity responses in E. carinicauda. These results indicated that E. carinicauda α-NAK involved in stress responses against salinity.

Whey peptides prevent chronic ultraviolet B radiation-induced skin aging in melanin-possessing male hairless mice.

Whey proteins or peptides exhibit various actions, including an antioxidant action, an anticancer action, and a protective action against childhood asthma and atopic syndrome. The effects of orally administered whey peptides (WPs) on chronic ultraviolet B (UVB) radiation-induced cutaneous changes, including changes in cutaneous thickness, elasticity, wrinkle formation, etc., have not been examined. In this study, we studied the preventive effects of WPs on cutaneous aging induced by chronic UVB irradiation in melanin-possessing male hairless mice (HRM). UVB (36-180 mJ/cm(2)) was irradiated to the dorsal area for 17 wk in HRM, and the measurements of cutaneous thickness and elasticity in UVB irradiated mice were performed every week. WPs (200 and 400 mg/kg, twice daily) were administered orally for 17 wk. WPs inhibited the increase in cutaneous thickness, wrinkle formation, and melanin granules and the reduction in cutaneous elasticity associated with photoaging. Furthermore, it has been reported that UVB irradiation-induced skin aging is closely associated with the increase in expression of matrix metalloproteinase (MMP), vascular endothelial growth factor (VEGF), Ki-67-, and 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive cells. WPs also prevented increases in the expression of MMP-2 and pro-MMP-9, VEGF, and Ki-67- and 8-OHdG-positive cells induced by chronic UVB irradiation. It was found that WPs prevent type IV collagen degradation, angiogenesis, proliferation, and DNA damage caused by UVB irradiation. Overall, these results demonstrate the considerable benefit of WPs for protection against solar UV-irradiated skin aging as a supplemental nutrient.

Fisetin inhibits matrix metalloproteinases and reduces tumor cell invasiveness and endothelial cell tube formation.

Matrix metalloproteinases (MMPs) play an important role in tissue remodeling during normal physiological situations and pathological implications such as tumor invasion and metastasis. MMP inhibitors were screened from extracts of medicinal herbs by an enzymatic assay using the MMP-14 catalytic domain. Among samples tested, a methanol extract of the root of Dalbergia odorifera T. CHEN (Leguminosae) showed the strongest inhibitory activity. The inhibitory component was purified through fractionation methods and identified as fisetin, abundant in many fruits and vegetables. In addition to inhibition of MMP-14, fisetin inhibits MMP-1, MMP-3, MMP-7, and MMP-9, more efficiently than a naturally occurring MMP inhibitor tetracycline. Fisetin dose-dependently inhibits proliferation of fibrosarcoma HT-1080 cells and human umbilical vascular endothelial cells (HUVECs), MMP-14-mediated activation of proMMP-2 in HT-1080 cells, invasiveness of HT-1080 cells, and in vitro tube formation of HUVECs. Therefore, fisetin could be valuable as a chemopreventive agent against cancer and a lead compound for development of therapeutic MMP inhibitors.

Cloning of prophenoloxidase from hemocytes of the blue crab, Callinectes sapidus and its expression and enzyme activity during the molt cycle.

The arthropods cuticle undergoes dramatic morphological and biochemical changes from being soft to hardness through each molting process. Prophenoloxidase (PPO) known as a key enzyme in the arthropod innate immune system involved in the melanization reaction, has been related with the initial shell-hardening process, specifically in the sclerotization of the protein matrix in the new cuticle. Since hemocytes have been reported as the main PPO source in arthropods, the transport of hemocyte PPO into the newly laid, soft cuticle has been proposed for shell-hardening occurring during and immediately after ecdysis. In order to define the role of hemocyte PPO in the shell-hardening of crustaceans, the full-length cDNA sequence (2806 nt) of hemocytes PPO of the blue crab Callinectes sapidus (CasPPO-hemo) is isolated using degenerate PCR and 5'-3' RACE. CasPPO-hemo encodes a putative PPO (672 aa) showing three hemocyanin domains: N, M, and C in order and two copper binding sites (CuA & CuB). The sequence analysis identifies the putative CasPPO-hemo as zymogen which requires the cleavage at the N-terminus for its activation. Hemocyte extract (CasHLS) contains the PO, the activity of which depends on the in vitro activation of trypsin. The expression levels of CasPPO-hemo are kept constant during the molt cycle. The increase in the number of hemocytes at early premolt correlates with the elevated PO activity, while at late premolt, the increment in hemocyte numbers does not reflect on the PO activity. The functional importance of the changes in the levels of CasHLS-PO activity during molt cycle is discussed in relation to cuticle hardening process.

Immune responses of prophenoloxidase and cytosolic manganese superoxide dismutase in the freshwater crayfish Cherax quadricarinatus against a virus and bacterium.

Prophenoloxidase (proPO) and cytosolic manganese superoxide dismutase (cytMnSOD) play crucial roles in crustacean innate immunity. In the present study, both of the above genes were cloned from hemocytes of the red claw crayfish Cherax quadricarinatus. A phylogenetic analysis of the amino acid sequences showed that C. quadricarinatus proPO and cytMnSOD were more closely related to the proPO and cytMnSOD of other crayfish than to those of penaeids, crabs, lobsters, or freshwater prawns. A tissue distribution analysis revealed that proPO was primarily expressed in hemocytes, gills, and the heart, while cytMnSOD was detected in all tissues examined. All of the crayfish artificially infected with white spot syndrome virus (WSSV) died within 4 days. According to a non-lethal dose, there was no mortality in crayfish when infected deliberately with Aeromonas hydrophila. Total hemocyte counts (THCs) had significantly decreased in crayfish at 48 and 72 h after infection with WSSV compared to the control group. In contrast, THCs of crayfish after A. hydrophila challenge had recovered by 48 and 72 h from a lower level at 24 h. There were similar responses in enzyme activities toward WSSV and A. hydrophila infection. Phenoloxidase (PO) and superoxide dismutase (SOD) activities per hemocyte significantly increased from 48 to 72 h compared to the control group. After WSSV challenge, expressions of proPO and cytMnSOD transcripts in hemocytes significantly decreased at 12h, then had respectively recovered and increased at 24 h. At 48-72 h, transcript levels were finally downregulated. No significant differences in the expression profiles of proPO and cytMnSOD were observed between the A. hydrophila-infected and control groups, besides the significant upregulation at 24h post-infection. These results implicate proPO and cytMnSOD in the immune response, and they presented similar expression patterns, although different defense mechanisms may exist for crayfish induced by WSSV and A. hydrophila.

Gene cloning and expression patterns of two prophenoloxidases from Catantops pinguis (Orthoptera: Catantopidae).

In insect, fat body plays major roles in insect innate immunity. Phenoloxidase (PO) is an important component in insect innate immunity and is necessary for acclimatization. In our study, two prophenoloxidase (PPO) subunits were obtained from fat body of Catantops pinguis (Stål). The full-length cDNA sequence of one PPO (CpPPO1) consisted of 2347 bp with an open reading frame (ORF) of 2187 bp encoding 728 amino acids, while the other subunit (CpPPO2) had a full length of 2445 bp, encoding 691 amino acids. Both the PPO gene products are predicted to possess all the structural features of other PPO members, including two putative tyrosinase copper-binding motifs with six highly conserved histidine residues and a thiolester-like motif. Tissue distribution analysis showed that both PPO mRNAs were abundantly expressed in the fat body among 11 tissues examined, and they were transiently up-regulated after Escherichia coli infection, consistent with them being immune-responsive genes. Total levels of CpPPO1 and CpPPO2 mRNA transcripts were much higher in first instar larvae and adults. A much higher transcript level of CpPPO1 was detected in several months, while there were extremely high mRNA expression levels of CpPPO2 in January, July, October, and December. The above results suggested that PPO from fat body might also bring significant function during the processes of development and acclimatization for C. pinguis.

A novel effect of imidazole derivative KK-42 on increasing survival of Aeromonas hydrophila challenged prawn Macrobrachium nipponense.

Imidazole derivative KK-42 is well known as the insect growth regulator. Here we find that KK-42 pretreatment could promote the survival of Macrobrachium nipponense infected with Aeromonas hydrophila, which is considered to be possibly related to the prophenoloxidase (proPO), a conserved copper-containing enzyme that plays an important role in defense against pathogens. In this study, a full-length of proPO gene from M. nipponense haemocytes, designated as MnproPO, was firstly cloned and characterized. The full-length cDNA contained 2428 bp with a 2013 bp open reading frame encoding a putative proPO protein of 671 amino acids with a predicted molecular mass of 76.5 kDa and pI of 7.31. It was predicted to possess all the expected features of proPO members, including two putative copper-binding sites with six histidine residues and a thiol ester-like motif. Sequence analysis showed that MnproPO exhibited the highest amino acid sequence similarity (93%) to a proPO of Macrobrachium rosenbergii. The gene was expressed highly in haemocytes and weakly in hepatopancreas. Real-time PCR analysis revealed that the MnproPO expression increased significantly at 3, 12 and 24 h after KK-42 treatment, the PO activity also importantly rose from 6 to 48 h in KK-42-treated prawns and reached the maximum at 24 h with a 2.3-fold higher than that in control group. Injection of A. hydrophila could stimulate the MnproPO transcription and PO activity whether or not the prawns were pretreated by KK-42, the mRNA level increased obviously only at 3 h and 6 h after the bacterium injection (challenged control), but increased constantly during the phase of experiment except at 6 h under the condition of KK-42 pretreatment (challenged treatment group). The change trend of PO activity was basically similar to that of MnproPO expression. Our present results demonstrate that the MnproPO expression as well as PO activity may be induced by KK-42, which is likely one of the molecular mechanisms of KK-42 acts for increasing survival of the prawn infected with A. hydrophila.

Molecular cloning of prophenoloxidase and the effects of dietary ß-glucan and rutin on immune response in hemocytes of the fleshy shrimp, Fenneropenaeus chinensis.

To investigate the effects of dietary ß-glucan (0.5 or 1 g kg⁻¹ diet: 0.5-BG, 1-BG) and rutin (0.5 or 1 g kg⁻¹ diet: 0.5-RT, 1-RT) after 10 days in the absence of pathogen challenge on the immune response of Fenneropenaeus chinensis, we determined total hemocyte count (THC) and the expression of four immune-related genes in hemocytes: those for prophenoloxidase (proPO), peroxinectin (PX), lipopolysaccharide and/or ß-glucan binding protein (LGBP), and c-type lectin (CL). As a prerequisite for subsequent experiments, cDNA encoding proPO of the fleshy shrimp, Fenneropenaeus chinensis (f-proPO) was obtained from hemocytes; it had a full length of 3023 bp, with an open reading frame (ORF) of 2061bp, a 105-bp 5'-untranslated region, and a 906-bp 3'-untranslated region containing the poly A signal. The THCs of shrimp fed ß-glucan of 1 g kg⁻¹ diet, and rutin of 1 g kg⁻¹ diet were significantly higher than that of the control (P < 0.05). The expression of proPO mRNA was slightly downregulated and that of LGBP mRNA was upregulated (except in 1-RT). PX and CL mRNA remained constitutively expressed in all groups. Our results reveal that ß-glucan and rutin dietary supplements have minimal effect on immune response in the absence of pathogen challenge.

Modulation of in vitro activity of zymogenic and mature recombinant human ß-secretase by dietary plants.

The in vitro activity of human recombinant ß-secretase (BACE1) was studied using a fluorogenic substrate based on the cleavage site for the enzyme in the Swedish mutation of amyloid precursor protein. The enzyme was inhibited by a control peptide inhibitor with good repeatability. The enzyme preparation comprised a mixture of pro-enzyme or zymogen and mature enzyme whereby the pro-enzyme sequence forms a 'flap' that can obstruct the binding site. 'Open flap' forms of the zymogen and mature enzyme are active, but the 'closed flap' form of the zymogen is inactive. This mixture of enzyme populations permitted apparent stimulation of enzyme activity under particular conditions, presumably due to facilitating flap-opening of the zymogen. As reported for heparin, enzyme activation was stimulated in the presence of low concentrations of Tween 20 and dimethylsulfoxide before becoming inhibited at higher concentrations. Dietary plant extracts either consistently inhibited (e.g. clove, tea, cinammon) or consistently stimulated (e.g. mushroom, parsley, asparagus) BACE1. Common structural features identified by Fourier transform infrared spectroscopy revealed that BACE1 activity could be explained by differential interactions of either small molecule or polymeric species with mature versus zymogen forms of the enzyme, respectively. Further, enzyme activity could be reversed by mixtures of high and low mass species. These results may have implications for the regulation of ß-secretase activity in vivo by either endogenous or possibly dietary factors and for a potential role of BACE1 in stimulation of the production of amyloid beta peptide in sporadic Alzheimer's disease.

A prophenoloxidase from Artemia sinica: cDNA cloning, expression and activity analysis during early development.

To understand the defense mechanisms of Crustacean animals, brine shrimp Artemia sinica prophenoloxidase (AsproPO) cDNA was cloned and its expression at early developmental stages was examined by reverse-transcription PCR (RT-PCR) and semi-quantitative RT-PCR, and activity of phenoloxidase (PO) at different developmental stages was further detected by using l-3,4-dihydroxyphenylalanine (l-DOPA) as a specific substrate in this study. It was found that the full-length of AsproPO cDNA is 2125 bp and it contains an open reading frame of 2100 bp encoding a protein of 699 amino acids. The deduced amino acid sequence of AsproPO has two putative copper binding sites highly conserved in Arthropods. Semi-quantitative RT-PCR analyses showed that the gene of AsproPO expressed at Emergence, Instar I and Instar II stages but did not at 0 h and 6 h stages. Activity measurement showed that PO activity could only be detected at Instar II stage but the other measured stages. All these implied that Artemia proPO immune system was complexly modulated during early development.

Intein lacking conserved C-terminal motif G retains controllable N-cleavage activity.

A split-intein consists of two complementary fragments (N-intein and C-intein) that can associate to carry out protein trans-splicing. The Ssp GyrB S11 split-intein is an engineered unconventional split-intein consisting of a 150-amino-acid N-intein and an extremely small six-amino-acid C-intein, which comprises the conserved intein motif G. Here, we show that fusion proteins containing the 150-amino-acid N-intein could be triggered to undergo controllable N-cleavage in vitro when the six-amino-acid C-intein or a derivative thereof was added as a synthetic peptide in trans. More importantly, we discovered, unexpectedly, that the 150-amino-acid N-intein could be induced by strong nucleophiles to undergo N-cleavage in vitro, and in Escherichia coli cells, in the absence of the motif G-containing six-amino-acid C-intein. This finding indicated that the first step of the protein splicing mechanism (acyl shift) could occur in the absence of the entire motif G. Extensive kinetic analyses revealed that both the motif G residues and the Ser+1 residue positively influenced N-cleavage rate constants and yields. The 150-amino-acid N-intein could also tolerate various unrelated sequences appended to its C-terminus without disruption of the N-cleavage function, suggesting that the catalytic pocket of the intein has considerable structural flexibility. Our findings reveal interesting insights into intein structure-function relationships, and demonstrate a new and potentially more useful method of controllable, intein-mediated N-cleavage for protein engineering applications.

Prophenoloxidase from Pieris rapae: gene cloning, activity, and transcription in response to venom/calyx fluid from the endoparasitoid wasp Cotesia glomerata.

Prophenoloxidase (PPO) plays an important role in melanization, necessary for defense against intruding parasitoids. Parasitoids have evolved to inject maternal virulence factors into the host hemocoel to suppress hemolymph melanization for the successful development of their progeny. In this study, the full-length complementary DNA (cDNA) of a Pieris rapae PPO was cloned. Its cDNA contained a 2 076-base pair (bp) open reading frame (ORF) encoding 691 amino acids (aa). Two putative copper-binding sites, a proteolytic activation site, three conserved hemocyanin domains, and a thiol ester motif were found in the deduced amino acid sequence. According to both multiple alignment and phylogenetic analysis, P. rapae PPO gene cloned here is a member of the lepidopteran PPO-2 family. Injection of Cotesia glomerata venom or calyx fluid resulted in reduction of P. rapae hemolymph phenoloxidase activity, demonstrating the ability to inhibit the host's melanization. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) showed that transcripts of P. rapae PPO-2 in the haemocytes from larvae had not significantly changed following venom injection, suggesting that the regulation of PPO messenger RNA (mRNA) expression by venom was not employed by C. glomerata to cause failure of melanization in parasitized host. While decreased P. rapae PPO-2 gene expression was observed in the haemocytes after calyx fluid injection, no detectable transcriptional change was induced by parasitization, indicating that transcriptional down-regulation of PPO by calyx fluid might play a minor role involved in inhibiting the host's melanization.

Molecular cloning and characterization of prophenoloxidase gene in swimming crab Portunus trituberculatus.

Phenoloxidase (PO), a melanin-synthesizing enzyme, found as a zymogen (proPO) in hemolymp, plays an important role in arthropod defence. In this study, a prophenoloxidase (proPO) cDNA was cloned from the haemocytes of swimming crab Portunus trituberculatus by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed that the cDNA clone had a full-length of 3040 bp, with an open reading frame of 2019 bp, a 5'-untranslated region of 138 bp, and a long 3'-untranslated region of 1707 bp. It encoded a protein of 672 amino acids which has a predicted molecular weight of 77.4 kDa and with an estimated pI of 6.19. It is predicted to possess all the expected features of proPO members, including two putative tyrosinase copper-binding motifs with six histidine residues and a proteolytic activation site. Comparison of amino acid sequences showed that the proPO-deduced amino acid of P. trituberculatus has an overall similarity of 56%-87% to that of other crustaceans. Northern blot analysis revealed that the presence of proPO was expressed in haemocytes, hepatopancreas and ovary, but not in eyestalk, gill and muscle. RT-PCR analysis indicated that proPO showed different expression profiles in crab haemocytes and hepatopancreas after Vibrio alginolyticus challenging. These facts indicated that proPO was potentially involved in the acute response against invading bacteria in P. trituberculatus.

A second proPO present in white shrimp Litopenaeus vannamei and expression of the proPOs during a Vibrio alginolyticus injection, molt stage, and oral sodium alginate ingestion.

Prophenoloxidase (proPO) is a melanin-synthesising enzyme that plays important roles in immune responses by crustaceans. Previously, we cloned and characterized proPO-I from white shrimp, Litopenaeus vannamei. In the present study, a novel prophenoloxidase-II (proPO-II) cDNA was also cloned from haemocytes of L. vannamei using oligonucleotide primers and reverse-transcriptase polymerase chain reaction (RT-PCR). Both 3'- and 5'-regions were isolated by the rapid amplification of complementary (c)DNA end (RACE) method. The 2504-bp cDNA contained an open reading frame (ORF) of 2073 bp, an 84-bp 5'-untranslated region, and a 347-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid sequence (691 amino acids) was 78.8 kDa with an estimated pI of 6.07. It contains two putative tyrosinase copper-binding motifs and a conserved C-terminal region common to all known proPOs. Comparisons of the amino acid sequences showed that white shrimp proPO-II is more closely related to the proPO of other penaeids than to that of crayfish, lobsters, crab, or a freshwater prawn, and is the ancestor type of known penaeid proPOs. proPO-I and proPO-II messenger (m)RNAs of shrimp were located on different loci, and were constitutively expressed mainly in haemocytes. The transcriptional regulation of these two proPOs in shrimp at different molt stages, those administered dietary sodium alginate, and those challenged with Vibrio alginolyticus were surveyed. The results showed that the proPOs may be directly involved in the acute-phase immune defence, and proPO-II may contribute earlier to immune defence in shrimp injected with V. alginolyticus, and it may be regulated by ecdysone. However, a similar effect was found by stimulating proPO-I and proPO-II mRNA expression in shrimp fed a sodium alginate-containing diet. Results of this study provide a basis for developing a comprehensive understanding of expression/function relationships of individual proPOs in shrimp.

[Isolation of a novel fructose-1,6-bisphosphate aldolase gene from Codonopsis lanceolata and analysis of the response of this gene to abiotic stresses].

A cDNA clone containing a fructose-1,6-bisphosphate aldolase (ALD) gene, designated ClAldC, was isolated from a medicinal plant Codonopsis lanceolata. ClAldC is predicted to encode a precursor protein of 358 amino acid residues, and its sequence shares high degrees of homology with a number of other ALDs. The expression of ClAldC in different C. lanceolata organs was analyzed using reverse transcriptase (RT)-PCR. The results showed that ClAldC expressed high in stems of intact plant, while expressed at low level in leaves and roots. In addition, the expression of ClAldC under different abiotic stresses was analyzed at different time points. Three tested abiotic stimuli, anoxygenic stress, hydrogen peroxide and chilling, triggered a significant induction of ClAldC within 2-8 h post-treatment. However, there was no induction under other four stresses, NaCI, wounding, light and dark. The positive responses of ClAldC to the three abiotic stimuli suggested that C. lanceolata ClAldC may help to protect against environmental stresses such as anoxia, chilling and oxidative stress.

A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: gene cloning, expression and activity analysis.

Prophenoloxidase (proPO) is a conserved copper-containing enzyme that plays important roles in immune response of crustaceans and insects. In the present study, the full-length cDNA of a prophenoloxidase (designated EsproPO) was cloned from haemocytes of Chinese mitten crab Eriocheir sinensis by expressed sequence tag (EST) and PCR techniques. The isolated 3549bp full-length cDNA of EsproPO contained a 2040bp open reading frame (ORF) encoding a putative proPO protein of 679 amino acids, a 5'-untranslated region (UTR) of 68bp, and a long 3'-UTR of 1441bp. Two putative copper-binding sites, a proteolytic activation site, and a complement-like motif (GCGWPQHM) were identified in the deduced amino acid sequence of EsproPO. Homology analysis revealed that EsproPO was highly similar to other proPOs from crustaceans with identities from 52% to 68%. The conserved domains and motifs, and higher similarity with other proPOs suggested that EsproPO was a member of the proPO family. The mRNA expression of EsproPO and PO specific activities in the tissues of hepatopancreas, gill, gonad, muscle, heart, eye and haemocytes were measured by quantitative real-time PCR and colorimetric assay, respectively. The mRNA transcripts of EsproPO and PO specific activities could be detected in all the examined tissues with the highest level both in hepatopancreas. Three peaks of EsproPO mRNA expression were recorded at 2h, 12h and 48h in haemocytes of Chinese mitten crab post Vibrio anguillarum challenge, which was consistent with the temporal profile of PO specific activity. The mRNA expression pattern and the activity fluctuation of EsproPO post V. anguillarum stimulation indicated that it was potentially involved in the acute response against invading bacteria in Chinese mitten crab.

Changes in immune gene expression and resistance to bacterial infection in lobster (Homarus gammarus) post-larval stage VI following acute or chronic exposure to immune stimulating compounds.

Real-time PCR was used to measure changes in transcript abundance of genes encoding important immune proteins, namely prophenoloxidase (proPO gene), beta-1,3-glucan binding protein (betaGBP gene) and a 12.2 kDa antimicrobial peptide (amp gene) in post-larval stage VI (PLVI) juveniles of the European lobster, Homarus gammarus. Gene expression was studied in both healthy PLVI and following single or repeat exposure to a range of compounds claimed to induce immune reactivity. A single acute (3-h) exposure to any of the tested stimulants did not produce a significant increase in expression of either the proPO or betaGBP genes, measured 6h after stimulation. However, there were a small sub-group of positive responders, identified mainly from betaGBP expression, within the experimental groups stimulated with either a beta-1,3-glucan or an alginate. There was also no significant increase in the expression of any of the three genes tested 24 h after repeated weekly (3-h) exposures to a either the beta-1,3-glucan or the alginate over the longer (36-day) period. The results do show that amp is expressed at an extremely high level compared to proPO or betaGBP in healthy animals and a significant correlation was found between the expression of proPO and both betaGBP and amp, irrespective of whether or not the larvae were stimulated. None of the immune stimulated compounds improved survival of PLVI challenged with the opportunistic pathogen, Listonella anguillarum, or the lobster pathogen, Aerococcus viridans var. homari. Thus, we found no evidence to support recent claims that immunity and disease resistance can be primed or promoted within a given population of crustaceans or that these animals exhibit functional immune memory to some soluble immune elicitors.